Mechanisms of action of the reverse transcriptase (RT) and protease of human immunodeficiency virus type 1 (HIV-1) are being investigated with the goal of developing specific inhibitors for these enzymes. i) Autoprocessing of the HIV-1 protease from a construct, MBP-TF-PR-Pol, containing the protease sequence (PR) flanked by a 19-amino acid P0l sequence at the C-terminus and a 12-amino acid sequence from the trans frame peptide (TF) at the N-terminus, fused to the maltose binding protein (MBP) of E. coli, involves two sequential proteolytic steps: cleavage at the N-terminus of the protease sequence to release a 13.2-kDa protein intermediate followed by C-terminal cleavage to give the mature, 11-kDa protease. The purified 13.2-kDa intermediate is virtually identical to the mature protease in its kinetic behavior, but it is somewhat more sensitive to denaturation by urea than the mature enzyme. There is no evidence for dissociation of the enzymatically active, dimeric form of this intermediate to inactive monomers at protein concentrations above 25 nM. Since the spontaneous conversion of the intermediate to the mature protease is second-order in protein concentration, its mechanism probably involves hydrolysis of the C- terminal fragment of one dimeric progein by a second dimer. ii) Kinetics of steady-state incorporation of a single nucleotide residue derived from thymidine or 4-thiothymidine triphosphate (TTP or 4S-TTP respectively) into a RNA-DNA template-primer by RT from HIV-1 were compared with those for the Klenow fragment of E. coli DNA polymerase I with a DNA template- primer. With the Klenow fragment, the Michaelis constant for 4S-TTP is 3.5 times larger than that for TTP, whereas with HIV-1 RT, the Michaelis constrants for the two triphosphates are essentially identical. With the Klenow fragment, the rate constant for turn-over of the enzyme is ca. 5 times larger with 4S-TTP than with its oxygen-containing analog, whereas with RT this rate constant for 4S-TTP is half that observd for TTP. The differences in turnover presumbly reflect differences in rates of dissociation of the oxygen- and sulfur-containing oligonucleotide products from the enzymes, since this step is likely to be rate- determining for the enzymatic reaction.